Spray-dried tetrasaccharides

ABSTRACT

Disclosed is a method for the manufacture of a spray-dried powder consisting essentially of LNT and/or LNnT, the spray-dried powder, its use for the manufacture of nutritional compositions, and nutritional compositions containing the spray-dried powder.

The present invention relates to preparations of human milkoligosaccharide. More specifically, the present invention relates tosolid preparations of human milk oligosaccharides and to methods for themanufacturing of said solid preparations of human milk oligosaccharides.

BACKGROUND

Human breast milk contains a substantial amount of carbohydrates. Thesecarbohydrates include monosaccharides such as L-fucose andN-acetylneuraminic acid (Neu5Ac). The disaccharide lactose is alsopresent in human breast milk. In addition to lactose, one liter of humanbreast milk contains up to 20 g/L oligosaccharides, the so-called “humanmilk oligosaccharides (HMOs)”. HMOs represent the third most abundantconstituent of human breast milk. It is presumed that more than 150structurally distinct oligosaccharides are present in human milk. Humanmilk usually contains between 10 and 13 major HMOs which are present ina concentration of between grams to several hundred milligrams per liter(Thurl et al., (2017), Nutrition Reviews 75(11) 920-933). HMOs includeneutral HMOs as well as acidic HMOs which contain one or more sialicacid moieties. The most prominent HMOs are shown in Table 1. Thestructural complexity and abundance of these oligosaccharides is uniquefor human milk and has not been found in the milk of other mammals suchas—for example—domesticated dairy animals.

Since HMOs are not digested by humans, the physiological role of thesesaccharides is under investigation for several decades. The prebioticeffect of HMOs has been discovered over 100 years ago. HMOs are able tomodulate the human gut microbiome by feeding beneficial bacteria.Several other functional effects of HMOs were investigated in the lastyears, especially their effect on neonates' development. HMOs are knownto act as decoys to reduce the risk of infections by bacterial and viralpathogens, which adhere to human cells by binding to cell surfaceglycoproteins. Additionally, various HMOs possess an anti-inflammatoryeffect and act as immunomodulators. Hence, it was proposed that HMOsreduce the risks of developing food allergies. A positive effect ofsialylated HMOs on neonatal brain development is intensely discussed(reviewed in “Prebiotics and Probiotics in Human Milk, Origins andfunctions of milk-borne oligosaccharides and bacteria”, Academic Press(2017) editors: McGuire M., McGuire M., and Bode L.).

Prominent HMOs are the tetraoses lacto-N-tetraose (LNT;Gal(β1,3)GlcNAc(β1,3) Gal(β1,4)Glc) and lacto-N-neotetraose (LNnT,Gal(β1,4)GlcNAc(β1,3)Gal(β1,4)Glc) which differ in the glycosidiclinkage of the terminal galactose moiety. Lacto-N-tetraose andlacto-N-neotetraose can be enzymatically synthesized by consecutiveadditions of GlcNAc and Gal residues to lactose. Lacto-N-tetraose andlacto-N-neotetraose are proposed to be substrates for the development ofthe intestinal microflora and the mucosal immune system.

A first step to take advantage of the beneficially effects of HMOs forbottle-fed infants is the addition of individual HMOs to infant formula.However, supplementing infant formulae with a combination ofstructurally distinct HMOs would be better, because a combination ofstructurally distinct HMOs will have effects that are more similar tothe effects of their original source, human milk, and which can not becaused by individual HMOs.

The limited supply of individual HMOs for supplementing infant formulaehas first led to the development of chemical syntheses of HMOs, followedby biocatalytic approaches using purified enzymes. Today, fermentationof genetically-engineered bacterial cells is used to produce differentHMOs in commercial scales (WO 2015/150328 A1, WO 2017/043382 A1, WO2010/070104 A1, WO 2012/097950 A1). The HMOs that are synthesized by thebacterial cells can be purified from the fermentation broth or celllysate to obtain substantially pure preparations of the HMOs such thatthey can be used in human food, especially in infant food.

During their purification, the LNT and/or LNnT is/are usually present inform of a liquid process stream. Along with the purification, theconcentration of LNT and/or LNnT in the process stream is increased.However, an aqueous solution of LNT and/or LNnT is very vulnerable forbacterial or fungal contamination. Therefore, it is preferred to providethe LNT and/or LNnT as a dry product having a low content of water suchthat microbial growth is impossible.

Typically, a saccharide is obtained in solid form by crystallization.Crystallization of individual HMOs has been described: for3-fucosyllactose (WO 2014/075680 A), for 2′-fucosyllactose (WO2011/150939 A), Di-fucosyllactose (WO 2016/086947 A), lacto-N-tetraose(WO 2017/101953 A), lacto-N-neotetraose (WO 2014/094783 A).Crystallization of HMOs involves the use of organic solvents such asalcohols, mainly ethanol or methanol, or organic acids such as glacialacetic acid. However, the use of organic solvents for crystallizing HMOsas last step in the process of obtaining the final product in solid formis not appropriate if the HMOs shall be used as food ingredients. Inaddition, organic solvents are harmful to the environment and to anyindividual handling them. Thus, the use of organic solvents requiresoccupational safety measures and appropriate disposal which renders theuse of organic solvents to be costly. Therefore, crystallization of HMOsto provide the HMOs in solid form has to be considered a drawback in theproduction of HMOs in an industrial scale.

Therefore, a process is desired which provides HMOs, in particular LNTand/or LNnT, in solid form, which is applicable in industrial scaleproduction of HMOs, and which does not involve the use of an organicsolvent at the end of the purification scheme to provide a solidpreparation of said HMO.

The problem is solved by a process for providing a powder consistingessentially of the purified HMO, wherein said method comprisesspray-drying of an aqueous solution which contains the HMO.

SUMMARY

In a first aspect, a spray-dried powder is provided method is providedconsisting essentially of one or more tetrasaccharides, preferably LNTand/or LNnT.

In a second aspect, a process for the manufacture of a spray-driedpowder consisting essentially of one or more tetrasaccharides,preferably LNT and/or LNnT.

In a third aspect, the use of the spray-dried powder consistingessentially of one or more tetrasaccharides, preferably LNT and/or LNnTfor the manufacture of a nutritional composition is provided.

In a fourth aspect, a nutritional composition containing the spray-driedpowder consisting essentially of one or more tetrasaccharides,preferably LNT and/or LNnT is provided.

DESCRIPTION OF THE FIGURE

FIG. 1 shows a graph illustrating the results of a X-ray powderdiffraction of spray-dried 3-fucosyllactose.

FIG. 2 shows a graph illustrating the results of a X-ray powderdiffraction of spray-dried lacto-N-tetraose.

FIG. 3 shows a graph illustrating the results of a X-ray powderdiffraction of spray-dried 6′-sialyllactose.

FIG. 4 shows a graph illustrating the results of a X-ray powderdiffraction of spray-dried 3′-sialyllactose.

FIG. 5 shows a graph illustrating the results of a X-ray powderdiffraction of a spray-dried mixture of 2′-fucosyllactose andlacto-N-tetraose.

FIG. 6 shows a graph illustrating the results of a X-ray powderdiffraction of a spray-dried mixture of 2′-fucosyllactose,3-fucosyllactose, lacto-N-tetraose, 3′-sialyl-lactose, and6′-sialyllactose.

DETAILED DESCRIPTION

According to the first aspect, a spray-dried powder consistingessentially of LNT and/or LNnT produced by microbial fermentation isprovided.

The LNT and/or LNnT is produced by microbial fermentation as describedherein below. The term “consisting essentially of” as used herein, meansthat the spray-dried powder consists of LNT and/or LNnTand—optionally—by-products that are generated during the microbialfermentation for the production of the LNT and/or LNnT, but which couldnot have been removed from a process stream obtained from the microbialfermentation. The term “consisting essentially of” includes spray-driedpowders consisting of at least 80%-wt., at least 85%-wt., at least90%-wt., at least 93%-wt., at least 95%-wt. or at least 98%-wt. LNTand/or LNnT.

In an additional and/or alternative embodiment, the LNT and/or LNnT ispresent in the spray-dried powder in amorphous form.

In an additional and/or alternative embodiment, the spray-dried powdercontains ≤15%-wt. of water, preferably ≤10%-wt. of water, morepreferably ≤7%-wt. of water, most preferably ≤5%-wt. of water.

In an additional and/or alternative embodiment, the spray-dried powderis free of genetically-engineered microorganisms and nucleic acidmolecules derived from genetically-engineered microorganisms.

According to the second aspect, provided is a process for themanufacture of a spray-dried powder consisting essentially of LNT and/orLNnT which has been produced by microbial fermentation. The processcomprises the steps of:

-   -   a) purifying LNT and/or LNnT from a fermentation broth;    -   b) providing an aqueous solution of the LNT and/or LNnT of step        a); and    -   c) subjecting the solution of step b) to spray-drying.

In an additional and/or alternative embodiment, purifying the LNT and/orLNnT from fermentation broth includes one or more of the steps of:

-   -   i) removing the microbial cells from the fermentation broth to        obtain a cleared process stream;    -   ii) subjecting the cleared process stream to at least one        ultrafiltration;    -   iii) treating the cleared process stream at least one time with        a cation exchange resin and/or at least one time with an anion        exchange resin;    -   iv) subjecting the cleared process stream to at least one        nanofiltration;    -   v) subjecting the cleared process stream to at least one        electrodialysis;    -   vi) treating the cleared process stream at least one time with        activated charcoal; and/or    -   vii) subjecting the cleared process stream at least one time to        a crystallization and/or precipitation step.

The LNT and/or LNnT may be produced by microbial fermentation, wherein agenetically-engineered microorganism that is able to synthesize LNTand/or LNnT is cultivated in a culture medium (fermentation broth) andunder conditions that are permissive for the synthesis of LNT and/orLNnT by said genetically-engineered microorganism. The purification ofLNT and/or LNnT produced by microbial fermentation comprises the step ofseparating the microbial cells from the fermentation broth to obtain acleared process stream which essentially free of cells and whichcontains the LNT and/or LNnT. This step is the first step in the processof purifying the desired oligosaccharides.

Suitable methods for separating the microbial cells from thefermentation broth include centrifugation wherein the microbial cellsare obtained as a pellet and the fermentation broth as a supernatant. Inan additional and/or alternative embodiment, the microbial cells areseparated from the fermentation broth by means of filtration. Suitablefiltration methods for separating the cells from the fermentation brothinclude microfiltration and ultrafiltration.

Microfiltration as such is a physical filtration process where aparticle-containing fluid is passed through a special pore-sizedmembrane to separate the particles from the fluid. The term“microfiltration” as used herein referst to a physical filtrationprocess where cells are seprated from the fermentation broth.

Ultrafiltration is a variety of membrane filtration and is notfundamentally different. In ultrafiltration, forces like pressure orconcentration gradients lead to a separation through a semipermeablemembrane. Cells, suspended solids and solutes of high molecular weightare retained in the so-called retentate, while water and low molecularweight solutes such as the desired sialylated oligosaccharide passthrough the membrane in the permeate (filtrate).

Ultrafiltration membranes are defined by the molecular weight cut-off(MWCO) of the membrane used. Ultrafiltration is applied in cross-flow ordead-end mode.

Typically, the microbial cells synthesize the LNT and/or LNnTintracellularly and secrete it into the fermentation broth. The thusproduced LNT and/or LNnT ends up in the fermentation broth which is thensubjected to further process steps for the purification of the LNTand/or LNnT as described herein after. Provided that the LNT and/or LNnTare present in the fermentation broth at the end of the fermentation,said fermentation broth becomes the cleared process stream after thecells have been removed. Provided that all or some of the LNT and/orLNnT remain intracellularly, the cells may be removed from thefrementation broth and lysed. Insoluble constituents may be removed fromthe cell lysate which then becomes the cleared process stream whichcontains the LNT and/or LNnT.

Notwithstanding that the process is used for the purification of LNTand/or LNnT that has been produced by microbial fermentation, saidprocess may also be employed to purify LNT and/or LNnT that was producedby enzymatic catalysis in-vitro. The LNT and/or LNnT can be purifiedfrom the reaction mixture at the end of the biocatalytic reaction. Saidreaction mixture is subjected to the process for the purification ascleared process stream.

The cleared process stream contains the LNT and/or LNnT as well as aby-products and undesired impurities such as—forexample—monosaccharides, disaccharides, undesired oligosaccharideby-products, ions, amino acids, polypeptides, proteins and/or nucleicacids.

In an additional and/or alternative embodiment, the process for thepurification of LNT and/or LNnT comprises the step of at least onecation exchange treatment to remove positively charged compounds fromthe cleared process stream.

Suitable cation exchange resins for removing postively charged compoundsinclude Lewatit S2568 (H+) (Lanxess AG, Cologne, DE).

In an additional and/or alternative embodiment, the process for thepurification of LNT and/or LNnT comprises the step of an anion exchangetreatment to remove undesired negatively charged compounds from thecleared process stream.

Suitable anion exchange resins include Lewatit S6368 A, Lewatit S4268,Lewatit S5528, Lewatit 56368A (Lanxess AG. Cologne, DE), Dowex AG 1×2(Mesh 200-400), Dowex 1×8 (Mesh 100-200), Purolite Chromalite CGA100×4(Purolite GmbH, Ratingen, DE), Dow Amberlite FPA51 (Dow Chemicals,Mich., USA).

In as additional/or alternative embodyment, the process for thepurification of LNT and/or LNnT comprises a nanofiltration and/or adiafiltration step to remove impurities having a lower molecular weight,and to concentrate the desired oligosaccharides.

Diafiltration involves the addition of fresh water to a solution inorder to remove (wash out) membrane-permeable components. Diafiltrationcan be used to separate components on the basis of their molecular sizeand charge by using appropriate membranes, wherein one or more speciesare efficiently retained and other species are membrane permeable. Inparticular, diafiltration using a nanofiltration membrane is effectivefor the separation of low molecular weight compounds like small moleculeand salts. Nanofiltration membranes usually have a molecular weightcut-off in the range 150-1000 Daltons. Nanofiltration is widely used inthe dairy industry for the concentration and demineralization of whey.

Suitable membranes for nanofiltration and/or diafiltration include DowFilmtec NF270-4040, Trisep 4040-XN45-TSF (Microdyn-Nadir GmbH,Wiesbaden, DE), GE4040F30 and GH4040F50 (GE Water & ProcessTechnologies, Ratingen, DE).

Diafiltration using nanofiltration membranes was found to be efficientas a pretreatment to remove significant amounts of contaminants prior toelectrodialysis treatment of the solution containing theoligosaccharide. The use of nanofiltration membranes for concentrationand diafiltration during the purification of HMOs results in lowerenergy and processing costs, and better product quality due to reducedthermal exposure, leading to reduced Maillard reactions and aldolreactions.

In an additional and/or alternative embodiment, the process for thepurification of LNT and/or LNnT comprises at least one electrodialysisstep.

Electrodialysis (ED) combines dialysis and electrolysis and can be usedfor the separation or concentration of ions in solutions based on theirselective electromigration through semipermeable membranes.

The basic principle of electrodialysis consists of an electrolytic cellcomprising a pair of electrodes submerged into an electrolyte for theconduction of ions, connected to a direct current generator. Theelectrode connected to the positive pole of the direct current generatoris the anode, and the electrode connected to the negative pole is thecathode. The electrolyte solution then supports the current flow, whichresults from the movement of negative and positive ions towards theanode and cathode, respectively. The membranes used for electrodialysisare essentially sheets of porous ion-exchange resins with negative orpositive charge groups, and are therefore described as cationic oranionic membranes, respectively. The ion-exchange membranes are usuallymade of polystyrene carrying a suitable functional group (such assulfonic acid for cationic membranes or a quaternary ammonium group foranionic membranes) cross-linked with divinylbenzene. The electrolyte canbe, for example, sodium chloride, sodium acetate, sodium propionate orsulfamic acid. The electrodialysis stack is then assembled in such a waythat the anionic and cationic membranes are parallel as in a filterpress between two electrode blocks, such that the stream undergoing iondepletion is well separated from the stream undergoing ion enrichment(the two solutions are also referred to as the diluate (undergoing iondepletion) and concentrate (undergoing ion enrichment). The heart of theelectrodialysis process is the membrane stack, which consists of severalanion-exchange membranes and cation-exchange membranes separated byspacers, installed between two electrodes. By applying a direct electriccurrent, anions and cations will migrate across the membranes towardsthe electrodes.

In an additional and/or alternative embodiment, the process for thepurification of LNT and/or LNnT further comprises a step of continuouschromatography like simulated bed moving (SMB) chromatography.

Simulated moving bed (SMB) chromatography originated in thepetrochemical and mineral industries. Today, SMB chromatography is usedby the pharmaceutical industry to isolate enantiomers from racemicmixtures. Large-scale SMB chromatography has already been used for theseparation of the monosaccharide fructose from fructose-glucosesolutions and for the separation of the disaccharide sucrose from sugarbeet or sugar cane syrups.

SMB processes used to separate saccharides use e.g. calcium charged,cross-linked polystyrene resins, anion resins in the bisulfite form(Bechthold M., et al., Chemie Ingenieur Technik, 2010, 82, 65-75), orpolystyrenic gel strong acid cation resin in the hydrogen form (PurolitePCR833H) (Purolite, Bala Cynwyd, USA).

Given the continuous mode of operation, the recycling of the mobilephase and also the potential to use large column sizes, SMB systems canin principle be scaled to achieve production volumes of hundreds oftons.

The process step of simulated moving bed chromatography is advantageousin that this process step allows further removal of oligosaccharidesbeing structurally closely related to the desired oligosaccharide.

In an additional and/or alternative embodiment, the process for thepurification of LNT and/or LNnT comprises a treatment of the processstream with activated charcoal to remove contaminating substances suchas colorants from the process stream.

In additional and/or alternative embodiment, the process for thepurification of LNT and/or LNnT comprises at least one step ofcrystallization or precipitation of LNT and/or LNnT from the clearedprocess stream. Crystallization or precipitation of LNT and/or LNnT fromthe process stream may be performed by adding a suitable amount of anorganic solvent that is miscible with water to the process streamcontaining the LNT and/or LNnT. The organic solvent may be selected fromthe group consisting of C₁- to C₆-alcohols and C₁- to C₄-carbon acids.

In an additional and/or alternative embodiment of the process for thepurification of the LNT and/or LNnT comprises a step sterile filtrationand/or endotoxin removal, preferably by filtration of the process streamthrough a 3 kDa filter or 6 kDa filter.

In an additional and/or alternative embodiment, the process for thepurification of LNT and/or LNnT comprises a step of increasing theconcentration of LNT and/or LNnT in the process stream. Theconcentration of LNT and/or LNnT in the process stream can be increasedby subjecting the process stream to vacuum evaporation, reverse osmosisor nanofiltration (e.g. nanofiltration with a nanofiltration membranehaving a size exclusion limit of ≤20 Å). Alternatively, crystallized orprecipitated LNT and/or LNnT is dissolved in water, to obtain a solutionof the LNT and/or LNnT possessing the desired concentration of LNTand/or LNnT.

In an additional and/or alternative embodiment, the resulting processstream is an aqueous solution which contains the LNT and/or LNnT in aconcentration of ≥20 g/L, ≥25 g/L, ≥30 g/L, ≥40 g/L, ≥60 g/L, ≥100 g/L,≥200 g/L or even ≥300 g/L.

In an additional and/or alternative embodiment, the aqueous solutioncontains the LNT and/or LNnT in a purity of at least 80%, at least 85%,at least 90%, at least 93%, at least 95% or at least 98% with respect tothe weight of dry matter/solutes within the solution.

The obtained concentrate containing the purified LNT and/or LNnT can bestored under appropriate conditions.

The process for the purification of LNT and/or LNnT is cost efficientand easy to scale up, making it suitable as a basis for a multi-tonscale manufacturing process.

The process for the purification of LNT and/or LNnT is also advantageousin that the aqueous solution is free of genetically-engineeredmicroorganisms and nucleic acid molecules derived fromgenetically-engineered microorganisms. In addition, the aqueous solutionis free of proteins. The total removal of proteins eliminates the riskof causing allergies to a potential consumer.

The process for the manufacture of the spray-dried powder comprises thestep of providing an aqueous solution containing the LNT and/or LNnT.

In an additional and/or alternative embodiment, the aqueous solutioncontains the LNT and/or LNnT in an amount of at least 20% (w/v), 30%(w/v), 35% (w/v), and up to 45% (w/v), 50% (w/v), 60% (w/v).

In an additional and/or alternative embodiment, the aqueous solutioncontains the LNT and/or LNnT in a purity of at least 80%, at least 85%,at least 90%, at least 93%, at least 95% or at least 98% with respect tothe weight of dry matter/solutes within the solution.

In an additional and/or alternative embodiment, the aqueous solutiondoes not contain genetically-engineered microorganisms, nucleic acidmolecules derived from genetically-engineered microorganisms andproteins.

In the process of the manufacture of the spray-dried powder, the aqueoussolution containing the LNT and/or LNnT is subjected to spray-drying.

Spray-drying is a method to obtain dry powders, wherein the solutioncontaining the substance of interest (i.e. LNT and/or LNnT) is firstsprayed into droplets which are rapidly dried by hot air. Spray-dryingis very fast and exposure of the substance to be dried to hightemperatures is quite short.

In an additional and/or alternative embodiment, the aqueous solutioncontaining the LNT and/or LNnT that has been purified from afermentation broth or process stream is spray-dried at a nozzletemperature of at least 110° C., preferably at least 120° C., morepreferably at least 125° C., and less than 150° C., preferably less than140° C. and more preferably less than 135° C.

In an additional and/or alternative embodiment, the aqueous solutioncontaining the LNT and/or LNnT that has been purified from afermentation broth or process stream is spray-dried at an outlettemperature of at least 60° C., preferably at least 65° C., and lessthan 80° C., preferably less than 70° C. In a particularly preferredembodiment, the aqueous solution containing the LNT and/or LNnT isspray-dried at a nozzle temperature of about 68° C. to about 70° C.

It is understood that LNT and LNnT can be purified and spray-driedindividually, and that the resulting spray-dried powders can be mixed inany desired ratio. In an additional and/or alternative embodiment,separate aqueous solutions containing LNT or LNnT respectively can bemixed in any desired ratio, and the resulting aqueous solutioncontaining LNT and LNnT in a desired ration can be subjected tospray-drying. The ratio of LNT and LNnT in the resulting spray-driedpowder corresponds to the ratio of LNT and LNnT in the aqueous solution.

The spray-drying of the aqueous solution containing 3-fucosyllactseprovides a powder of low hygroscopy, wherein the LNT and/or LNnT ispresent in amorphous form, and wherein the particle size is homogeneous.

According to the third aspect, provided is the use of the spray-driedpowder containing LNT and/or LNnT that has been purified from a processstream for the manufacture of a nutritional composition. The spray-driedpowder consisting essentially of LNT and/or LNnT is suitable for humanconsumptions and may thus be included into preparations for humanconsumption such as medicinal formulations, infant formula, dairy drinksor dietary supplements.

According to the fourth aspect, provided are nutritional compositionswhich contain a spray-dried powder as described in the first aspect ofas manufactured according to the second aspect.

In an additional and/or alternative embodiment, the nutritionalcomposition contains at least one additional HMO which is not LNT and/orLNnT. The at least one additional HMO may be a neutral HMO, preferablyselected from the group consisting of 2′-fucosyllactose (2′-FL),3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose(LNnT), and lacto-N-fucopentaose I (LNFPI). In an additional and/oralternative embodiment, the at least one additional HMO may be asialylated HMO, preferably selected from the group consisting of3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL),sialyllacto-N-tetraose (LST)-a, LST-b, LST-c anddisialyllacto-N-tetraose (DSLNT).

TABLE 1 Composition of an exemplary mixture containing suitable assupplement for infant formulae. Proportion in mix (percentage by Finalconcentration in infant Compound weight) formula (g/L) 2′-FL 34 2.5 3-FL11 0.8 LNT 20 1.5 LNnT 2 0.15 LNFPI 13 1.0 3′-SL 3 0.2 6′-SL 4 0.3Neu5Ac 8 0.6 L-Fucose 5 0.4 Total 100 7.45

In an additional and/or alternative embodiment, the nutritionalcomposition includes a mixture consisting essentially of Neu5Ac, 2′-FL,3-FL, LNT, LNnT, LNFPI, 3′-SL, 6′-SL, sialic acid and L-fucose. Anutritional composition including preferred amounts of each of saidcompounds is provided in Table 1.

The composition according to the second column in Table 1 is ofparticular advantage for supplementing infant formula such that thefinal infant formula for direct consumption may contain the compounds ofthe mixture in concentrations as specified in the third column of Table1.

In an additional and/or alternative embodiment, the nutritionalcomposition contains one or more additional ingredients. Said one ormore additional ingredients are selected from the group consisting ofoil, fat and fatty acids (such as olive oil, sunflower oil, coconut oil,nut oil, rapeseed oil, palm oil, flaxseed oil, fish oil, linolenic acid,soy-bean oil, etc.), carbohydrates (such as glucose, fructose, lactose,maltodextrin, starch, sucrose, inositol, etc.) proteins (from skim milk,whey, casein (derived from any domesticated dairy animals), or soybean), vitamins (A, B1, B2, B5, B6, B12,C, D, E, K, biotin, folic acid,niacin, choline) minerals and trace elements (sodium, potassium,chloride, calcium, phosphorus, magnesium, iron, zinc, manganese,fluoride, selenium, iodine, copper).

In a preferred embodiment the nutritional composition containing thespray dried human milk oligosaccharides or the mixture of human milkoligosaccharides or the mixture of human milk oligosaccharides withfunctional monosaccharides or the mixture of human milk oligosaccharideswith other fibers is an infant formula that meets the compositionalrequirements set forth in Regulation (EU) 2016/127 and/or in the Code ofFederal Regulations (USA) Title 21 107.100 (nutrient specifications).Representative compositions of infant formulas are specified in Tables 2and 3.

TABLE 2 Components of an exemplary infant formula. Infant formula:Skimmed milk Vegetable oils (palm oil, rapeseed oil, sunflower oil)Human milk oligosaccharides LNT and/or LNnT Skimmed milk powder Oil ofMortierella alpine Fish oil Calcium carbonate Potassium chloride VitaminC Sodium chloride Vitamin E Iron acetate Zinc sulfate NiacinCalcium-D-panthothenate Copper sulfate Vitamin A Vitamin B1 Vitamin B6Magnesium sulfate Potassium iodate Folic acid Vitamin K Sodium seleniteVitamin D

TABLE 3 Composition of an exemplary infant formula. The finalconcentration is based on a preparation of 13.5 g of the powder in 90 mlof water. per 100 ml per 100 g powder infant formula Energy kJ 2094-2145283    kcal 500-512 67-68 Fats, hereof: g 24.2-26.2 3.3-3.5 saturatedfatty acids g 8.7-9.4 1.2-1.3 monounsaturated fatty acids g 10.4  1.4 polyunsaturated fatty acids g 5.5-5.9 0.7-0.8 Carbohydrates hereof: g56-58 7.4-7.9 Sugars g 44-56  6-7.4 hereof: Lactose g 44-56  6-7.4Neu5Ac mg 440    60    L-fucose mg 300    40    HMOs hereof g 4.22-4.810.57-0.65 2′-FL g 1.85-2.22 0.25-0.30 3-FL mg 555.56-592.6  75-80 LNT g1.11 0.15 LNnT mg    0-111.11  0-15 LNPF-I mg    0-740.74  0-100 3′-SLmg 148.15-170.37 20-23 6′-SL mg  207.4-222.22 28-30 Protein g11.11-11.85 1.5-1.6 Salt g 0.47-0.59 0.06-0.08 Vitamins Vitamin A μg357-358 47.3-48.2 Vitamin D μg 7.8  1.05 Vitamin E mg 8.15 1.1  VitaminK μg 43.7-44.4 5.9-6.0 Vitamin C mg 115-118 15-16 Vitamin B1 mg0.51-0.60 0.068-0.079 Vitamin B2 mg 1.3-1.7 0.18-0.23 Niacin mg 3.630.49 Vitamin B6 μg 526-600 71-81 Folic acid μg 160-164 21.6-21.7 VitaminB12 μg 1.7-1.9 0.23-0.25 Biotin μg 22-30 3.0-3.9 Panthothenic acid mg4.6-5.4 0.62-0.72 Minerals Sodium mg 187-236 25.3-31.2 Potassium mg673-675 88.8-91.2 Chloride mg 327-333 43.1-44.9 Calcium mg 460-50462.1-66.5 Phosphorous mg 335-352 45.2-46.5 Magnesium mg 49.3-56.36.66-7.43 Iron mg 4.15 0.56 Zinc mg 3.7-3.8 0.49-0.51 Copper μg 274   37    Manganese μg 96.3  13    Fluoride μg 30.4-32.6 4.1-4.4 Selenium μg11.1-12.3 1.5-1.6 Iodine μg 101.5-103.7 13.7-14 

In an additional and/or alternative embodiment, the nutritionalcomposition also contains microorganisms, preferably probioticmicroorganisms. For infant food applications, the preferredmicroorganisms are derived from or can be found in the microbiome of ahealthy human. Preferably, but with no limitations, the microorganismsare selected from the genera Bifidobacterium, Lactobacillus,Enterococcus, Streptococcus, Staphylococcus, Peptostreptococcus,Leuconostoc, Clostridium, Eubacterium, Veilonella, Fusobacterium,Bacterioides, Prevotella, Escherichia, Propionibacterium andSaccharomyces. In an additional and/or alternative embodiment, themicroorganism is selected from the group consisting of Bifidobacteriumadolescentis, B. animalis, B. bifidum, B. breve, B. infantis, B. lactis,B. longum; Enterococcus faecium; Escherichia coli; Klyveromycesmarxianus; Lactobacillus acidophilus, L. bulgaricus, L. casei, L.crispatus, L. fermentum, L. gasseri, L. helveticus, L. johnsonii, L.paracasei, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius, L.sakei; Lactococcus lactis (including but not limited to the subspecieslactis, cremoris and diacetylactis); Leuconostoc mesenteroides(including but not limited to subspecies mesenteroides); Pedicoccusacidilactici, P. pentosaceus; Propionibacterium acidipropionici, P.freudenreichii ssp. shermanii; Staphylococcus carnosus; andStreptococcus thermophilus.

In addition to the combination living organisms, the nutritionalcomposition can also include dead cell cultures. In the field ofprobiotics, killed cell cultures are sometimes used (e.g. tyndalizedbacteria). These killed cultures may provide proteins, peptides,oligosaccharides, cell outer wall fragments and natural products,leading to the short-term stimulation of the immune system.

Including probiotic microorganisms in the nutritional composition,especially in the presence of HMOs, is particularly advantageous in thatit also promotes the establishment of a healthy gut microbiome.

In an additional and/or alternative embodiment, the nutritionalcomposition also includes prebiotics such as galacto-oligosaccharides(GOS), fructo-oligosaccharides (FOS), inulin or combinations thereof.

The nutritional composition is present in solid form including, but notlimited to, powders, granules, flakes, pellets or combinations thereof.

In an additional embodiment, the nutritional composition is selectedfrom the group consisting of medicinal formulations, infant formulas,dairy drinks and dietary supplements.

As a medicinal formulation, the nutritional composition may be used toimprove cognitive performance, especially for improving attention,learning and/or memory.

The present invention will be described with respect to particularembodiments and with reference to drawings, but the invention is notlimited thereto but only by the claims. Furthermore, the terms first,second and the like in the description and in the claims, are used fordistinguishing between similar elements and not necessarily fordescribing a sequence, either temporally, spatially, in ranking or inany other manner. It is to be understood that the terms so used areinterchangeable under appropriate circumstances and that the embodimentsof the invention described herein are capable of operation in othersequences than described or illustrated herein.

It is to be noticed that the term “comprising”, as used in the claims,should not be interpreted as being restricted to the means listedthereafter; it does not exclude other elements or steps. It is thus tobe interpreted as specifying the presence of the stated features,integers, steps or components as referred to, but does not preclude thepresence or addition of one or more other features, integers, steps orcomponents, or groups thereof. Thus, the scope of the expression “adevice comprising means A and B” should not be limited to devicesconsisting only of components A and B. It means that with respect to thepresent invention, the only relevant components of the device are A andB.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, appearances of the phrases“in one embodiment” or “in an embodiment” in various places throughoutthis specification do not necessarily refer always to the sameembodiment. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would beapparent to one of ordinary skill in the art from this disclosure, inone or more embodiments.

Similarly, it should be appreciated that in the description ofrepresentative embodiments of the invention, various features of theinvention are sometimes grouped together in a single embodiment, figure,or description thereof for the purpose of streamlining the disclosureand facilitating the understanding of one or more of the variousinventive aspects. This method of disclosure is not to be interpreted asreflecting an intention that the claimed invention requires morefeatures than are expressly listed in each claim. Rather, as thefollowing claims reflect, inventive aspects may require fewer than allthe features of any foregoing disclosed embodiment. Thus, the claimsfollowing the detailed description are hereby expressly incorporatedinto this detailed description, with each claim standing on its own as aseparate embodiment of this invention.

Furthermore, whereas some embodiments described herein include some butnot other features included in other embodiments, combinations offeatures of different embodiments are meant to be within the scope ofthe invention, and form different embodiments, as would be understood bythose familiar with the art. For example, in the following claims, anyof the claimed embodiments can be used in any combination.

Furthermore, some of the embodiments are described herein as a method orcombination of elements of a method that can be implemented by aprocessor of a computer system or by other means of carrying out thefunction. Thus, a processor with the necessary instructions for carryingout such a method or element of a method forms a means for carrying outthe method or element of a method. Furthermore, an element describedherein of an apparatus embodiment is an example of a means for carryingout the function performed by the element for the purpose of carryingout the invention.

In the description and drawings provided herein, numerous specificdetails are set forth. However, it is understood that embodiments of theinvention may be practiced without these specific details. In otherinstances, well-known methods, structures and techniques have not beenshown in detail in order to facilitate the understanding of thedescription and drawings.

The invention will now be described by a detailed description of severalembodiments of the invention. It is clear that other embodiments of theinvention can be configured according to the knowledge of personsskilled in the art without departing from the true spirit or technicalmerit of the invention, the invention being limited only by the terms ofthe appended claims.

EXAMPLE 1 Purification of T-Fucosyllactose from Fermentation Broth

Production of 2′-fucosyllactose by fermentation using a geneticallymodified E. coli strain was performed as described in European patentapplication No. 16 196 486.1. The 2′-fucosyllactose was purified fromthe fermentation broth by filtration, ion exchange chromatography,nanofiltration, diafiltration or electrodialysis, and treatment withcharcoal as described in WO 2015/106943 A1. The resulting solutioncontaining 2′-fucosyllactose was subjected to spray-drying to obtain astable solid product.

EXAMPLE 2 Purification of 3-Fucosyllactose from Fermentation Broth

3-Fucosyllactose was produced by fermentation using a geneticallymodified E. coli strain as described in European patent application No.16 196 486.1.

The cells were separated from the culture medium by ultrafiltration(0.05 μm cut-off) (CUT membrane technology, Erkrath, Germany) followedby a cross-flow filter with a MWCO of 150 kDa (Microdyn-Nadir,Wiesbaden, Germany). The cell-free fermentation medium containing about30 g/L 3-fucosyllactose, was passed over a strong cationic ion exchanger(Lewatit S 2568 (Lanxess, Cologne, Germany) in H⁺ form to removepositive charged contaminants. Afterwards the solution was set to pH 7.0using sodium hydroxide and applied to an anionic ion exchanger (LewatitS6368 A, Lanxess) in the chloride form. Both ion exchangers were used in200 L volume. After a second filtration (150 kDa; Microdyn-Nadir,Wiesbaden, Germany) the particle free solution was concentrated 5-foldby nanofiltration using a Filmtech NF270 membrane (Dow, Midland, USA)and 2.5-fold by vacuum evaporation. The concentrated solution and aconductivity of about 15 mS cm⁻¹ was filtrated (10 kDa; Microdyn-Nadir,Wiesbaden, Germany), clarified by activated carbon charcoal(CAS:7440-44-0, Carl Roth, Karlsruhe, Germany) and deionized byelectrodialysis. Therefor a PC-Cell BED 1-3 electrodialysis apparatus(PC-Cell, Heusweiler, Germany) with a PC-Cell E200 membrane stack wasused containing the following membranes: cation exchange membrane CEM:PCSK and anion membrane AEM:PCAcid60. 0.25 M Sulphamic acid was used aselectrolyte in the process. For reduction of brownish coloring caused byMaillard-reactions and aldol products originating from the fermentationprocess, a second round of ion exchange chromatography was performedusing the same ion exchange material as aforementioned in Na⁺ and Cl⁻form, however in a volume of 50 L. After concentrating the sugarsolution by evaporation, again the conductivity was reduced from 4 mScm⁻¹ to 0.4 mS cm⁻¹ or less by electrodialysis using the PC-Cell BED 1-3mentioned previously. For further decolorization the solution was mixedwith activated charcoal (CAS:7440-44-0, Carl Roth, Karlsruhe, Germany)and a nearly colorless solution was obtained by filtration.

EXAMPLE 3 Purification of Lacto-N-Tetraose from Fermentation Broth

Fermentative production of Lacto-N-tetraose was conducted using agenetically modified E. coli BL21 (DE3) ΔlacZ strain, with genomicallyintegrated genes essential for the in vivo synthesis ofLacto-N-tetraose., namely, a N-acetylglucosamine glycosyltransferase(lgtA from Neisseria meningitidis MC58), a β-1,3-galactosyltransferases(wbdO from Salmonella enterica subsp. salamae serovar Greenside), lacYfrom E. coli K12, the UDP-glucose-4-epimerase galE, and theUTP-glucose-1-phosphat uridyltransferase galU, both from E. coli K12. Inaddition, the galS gene encoding a glucosamine-6-phosphate synthase wasoverexpressed. For the fermentative production of Lacto-N-tetraose thestrain was grown in a defined mineral salts medium comprising 2% glucoseas carbon source. Antifoam was added when needed. The pH was controlledusing a 25% ammonia solution. Lactose was added stepwise to a finalconcentration of 15 mM from a 216 g l⁻¹ lactose stock, the lactoseconcentration in the culture medium was held constant throw-out thefermentation process. Residual lactose and Lacto-N-triose II,accumulation during the process as by-product, was hydrolyzed by asecond E. coli strain that was added to the fermenter. This strainexpressed a functional beta-lactamase, a beta-N-acetylhexosaminidase(bbhl from Bifidobacterium bifidum JCM1254), and a functional gal-operonfor degradation of monosaccharides (EP 2 845 905 A).

Cells were separated from the fermentation broth, and thelacto-N-tetraose containing fluid was purified to a purity of 75-80%,determined by mass balance, according to the procedure described inexample 2.

Contaminating carbohydrate by-products resulting from inefficientenzymatic degradation and metabolization were removed by chromatographyusing a simulated moving bed (SMB) chromatography, according to WO2015/049331. Alternatively, the lacto-N-tetraose was purified bycrystallization with isopropanol. For crystallization thelacto-N-tetraose containing solution was concentrated by evaporation toa concentration of 20% and spray-dried. Using a NUBILOSA LTC-GMP spraydryer (NUBILOSA, Konstanz, Germany) the solution was passed undernitrogen flow through the spray dryers nozzles with an inlet temperatureof 130° C. while the product flow was controlled to maintain an outlettemperature of 67° C. to 68° C.

The solid material was added to a mixture of isopropanol and water (3:1(vol/vol)) in a ratio of 1 kg powder in 12 L isopropanol/water. Thesuspension was stirred vigorously, then the insoluble lacto-N-tetraosewas filtrated and dried at 40° C. Starting with a 73-89% pure material,the crystallized Lacto-N-tetraose was purified to about 95%, with arecovery of 85%. The sugar was dissolved in water to a concentration of25% and passed sequentially through a 6 kDa filter (Pall Microzaultrafiltration module SIP-2013, Pall Corporation, Dreieich, Germany)and a 0.2 μm sterile filter. Solid material was obtained by spray dryingthe sterile material under the conditions described above.

EXAMPLE 4 Purification of 3′- and 6′-Sialyllactose from FermentationBroth

For production of 3′- sialyllactose and 6′-sialyllactose recombinant E.coli BL21 (DE3) ΔlacZ strains were used. The strains had common geneticmodifications: chromosomal, constitutive expression of theglucosamine-6-phosphate synthase GlmS from E. coli, theN-acetylglucosamin2-epimerase Slr1975 from Synechocystis sp., theglucosamine 6-phosphat N-acetyltransferase Gna1 from Saccharomycescerevisiae, the phosphoenolpyruvate synthase PpsA from E. coli, theN-acetylneura-minate synthase NeuB, and the CMP-sialic acid synthetaseNeuA, the latter both from Campylobacter jejuni. Additionally, the genesencoding the lactose permease LacY from E. coli, cscB (sucrosepermease), cscK (fructokinase), cscA (sucrose hydrolase), and cscR(transcriptional regulator) from E. coli W, and a functional gal-operon,consisting of the genes galE (UDP-glucose-4-epimerase), galT(galactose-1-phosphate uridylyltransferase), galK (galactokinase), andgalM (galactose-1-epimerase) from E. coli K12 were integrated into thegenome of the BL21 strain, and constitutively expressed.

The strain synthesizing 3′-sialyllactose harbors thealpha-2,3-sialyltransferase gene from Vibrio sp. JT-FAJ-16, while the6′-sialyllactose producing strain contains thealpha-2,6-sialyltransferase plsT6 from Photobacterium leiognathiJT-SHIZ-119.

The sialyllactose producing strains were grown in a defined mineralsalts medium containing 2% sucrose as carbon source. The sucrose feed(500 g l⁻¹), fed in the fed-batch phase, was supplemented with 8 mMMgSO₄, 0.1 mM CaCl₂, trace elements, and 5 g l⁻¹ NH₄Cl.

For sialyllactose formation, a lactose feed of 216 g l⁻¹ was employed.The pH was controlled by using ammonia solution (25% v/v). Fed batchfermentation was conducted at 30° C. under constant aeration andagitation. In order to remove residual lactose at the end of thefermentation, β-galactosidase was added to the fermentation vessel. Theresulting monosaccharides were metabolized by the production strain.

The cell-free liquid was then deionized by ion exchange chromatography.First, cationic contaminants were removed on a strong cationic exchangerin a volume of 200 L (Lewatit S 2568 (Lanxess, Cologne, Germany) in H⁺form. Using NaOH the pH of the obtained solution was set to 7.0. In asecond step, anionic ions and undesired colorants were removed from thesolution using the strong anionic exchanger Lewatit S 6368 S (Lanxess,Cologne, Germany) in the chloride form. The ion exchanger had a bedvolume of 200 L. Using a second filtration step on the cross-flow filter(150 kDa cut-off) (Microdyn-Nadir, Wiesbaden, Germany), precipitatesoriginating from acidifying the solution were removed. For concentrationof the sugar, the solution was nanofiltrated on a Dow FILMTECHNF270-4040 (Inagua, Mönchengladbach, Germany), or, alternatively on aTrisep 4040-XN45-TSF Membrane (0.5 kDa cut-off) (Microdyn-Nadir,Wiesbaden, Germany). Using the latter, the monosaccharideN-acetylglucosamine, originating from the fermentation process andcontaminating the sialyllactose solution, was separated from theproduct. The concentrated sialyllactose solution was then treated withactivated charcoal (CAS:7440-44-0, Carl Roth, Karlsruhe, Germany) toremove colorants such as Maillard reaction products and aldol reactionproducts. In order to separate the sialyllactose from by-products thatoriginate from the fermentation process like sialic acid andN-acetylglucosmine, the solution was filtrated on with a 1 kDa cut-offmembrane GE4040F30 (GE water & process technologies, Ratingen, Germany),and diafiltrated to a conductivity of 0.6 to 0.8 mS cm⁻¹. The dilutedsolution was concentrated on a rotary evaporator to a concentration ofabout 300 g/L. In a final chromatographic separation other contaminatingsugars, like di-sialyllactose were removed. Therefor the concentratedsolution was applied to a weak anion ion exchange resin in the acetateform (Amberlite FPA51, Dow Chemical, Michigan, USA). While thesialyllactose rarely binds to the resin, the di-sialyllactose isadsorbed. Thus, the sialyllactose is eluted with 10 mM ammoniumacetat,while the di-sialyllactose is eluted with 1 M ammoniumacetat. Forremoval of the ammoniumacetat, the sialyllactose was precipitated with a10-fold excess of ethanol. The solid fraction was filtrated and dried.

The product was finalized by passing a 20% sialyllactose solutionsequentially through a 6 kDa filter (Pall Microza ultrafiltration moduleSIP-2013, Pall Corporation, Dreieich, Germany) and a 0.2 μm sterilefilter.

A part of the solution was spray dried using a Büchi spray dryer (BüchiMini Spray Dryer B-290) (Büchi, Essen, Germany), applying the followingparameters: Inlet-temperature: 130° C., Outlet temperature 67° C.-71°C., gasflow 670 L/h, aspirator 100%.

The spray-dried 6′-sialyllactose had a purity of 91%, while the3′-sialyllactose material had a purity of 93%.

EXAMPLE 5 Preparations of HMO Mixtures

Mixtures of HMOs were prepared from solid products. Therefor the singleHMOs were spray-dried and the powder were mixed. HMO-Mix I contained2′-fucosyllactose and lacto-N-tetraose in a ratio of 70% to 30%; HMO-MixII contained 2′-fucosyllactose (52%), 3-fucosyllactose (13%),lacto-N-tetraose (26%), 3′-sialyllactose (4%), and 6′-sialyllactose(5%). The mixed powders were solved in water to a solution of 20% sugar,and spray-died again using the Büchi spray dryer as described in example4.

EXAMPLE 6 Characterisation of Spray-Dried Human Milk Oligosaccharides

6.1 Differential Scanning Calorimetry (DSC)

Using differential scanning calorimetry (DSC) on a Mettler Toledo 821e(Mettler Toledo, Giessen, Germany) thermal events of spray-dried humanmilk oligosaccharides, namely 3-fucosyllactose, 6′-sialyllactose,3′-sialyllactose, lacto-N-tetraose, and spray-dried mixtures of humanmilk oligosaccharides, a mixture (HMO-Mix I) of2′-fucosyllactose/lacto-N-tetraose, and a mixture (HMO Mix II) of2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 6′-sialyllactose,3′-sialyllactose, respectively, were determined.

A Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) was used todetermine thermal events of the spray-dried products (glass transitiontemperature (Tg), further exo- and endothermic events).

Approximately 25 mg of the spray-dried human milk oligosaccharides wereanalyzed in crimped Al-crucibles (Mettler Toledo, Giessen, Germany). Thesamples were cooled to 0° C. with 10 K/min and reheated to 100° C. witha scanning rate of 10 K/min. After cooling down the samples to 0° C. ina second heating cycle, the samples were reheated to 150° C. Themidpoint of the endothermic shift of the baseline during the heatingscan was taken as glass transition temperature (Tg). Exothermic andendothermic peaks are reported by means of the peak temperature and thenormalized energy of the event.

The first heating scan in all samples showed a main glass transitionevent in the total heat flow, as evidenced by a main step transition inthe range of approximately 48-58° C., in most of the samples, the majorglass transition event observed in the first heating scan reoccurred inthe second heating scan. The results of the DSC analyses are summarizedin table 4.

TABLE 4 Thermal events of HMOs as determined by differential scanningcalorimetry 1^(st) heating scan 2^(nd) heating scan Sample Tg [° C.] Tg[° C.] 3-fucosyllactose 57.6 59.9 lacto-N-tetraose 49.9 79.46′-sialyllactose 47.6 49.6 3′-sialyllactose 48.8 54.32′-fucosyllactose/lacto-N-tetraose 56.3 59 HMO Mix 54.2 55.6

For 3-fucosyllactose an endothermal relaxation peak after Tg in thefirst heating scan was detected. For lacto-N-tetraose a much higher Tgof about 79° C. was detected in the 2^(nd) heating scan compared to thatof the other samples. This might be caused by an endothermal eventduring the first heating scan at about 89° C. (−6.04 J/g). Like for3-fucosyllactose, also for 6′-sialyllactose an endothermal relaxationpeak was detected after Tg, however, in this sample additionally anendothermal event occurred at 77° C. (−0.22 J/g). No endothermal eventswere detected for the 3′-sialyllactose and the HMO-Mix I, for HMO-Mix IIthe endothermal event during the 1^(st) heating scan was at 79° C. (0.34J/g).

6.2 X-Ray Powder Diffraction (XRD)

Wide angle X-ray powder diffraction (XRD) was used to study themorphology of lyophilized products. The X-ray diffractometer Empyrean(Panalytical, Almelo, The Netherlands) equipped with a copper anode (45kV, 40 mA, K_(α1) emission at a wavelength of 0.154 nm) and a PIXcel3Ddetector was used. Approximately 100 mg the spray-dried samples wereanalyzed in reflection mode in the angular range from 5-45° 2θ, with astep size of 0.04° 2θ and a counting time of 100 seconds per step.

All singular oligosaccharides as well as the HMO Mixes I and II showed afully amorphous state (FIGS. 1 to 6). For lacto-N-tetraose a second(amorphous) signal was detected around 9-10°.

6.3 Laser Diffraction

The powder particle size was assessed by laser diffraction. The systemdetects scattered and diffracted light by an array of concentricallyarranged sensor elements. The software-algorithm is then approximatingthe particle counts by calculating the z-values of the light intensityvalues, which arrive at the different sensor elements. The analysis wasexecuted using a SALD-7500 Aggregate Sizer (Shimadzu Corporation, Kyoto,Japan) quantitative laser diffraction system (qLD).

A small amount (spatula tip) of each sample was dispersed in 2 mlisooctane and homogenized by ultrasonication for five minutes. Thedispersion was transferred into a batch cell filled with isooctane andanalyzed in manual mode.

Data acquisition settings were as follows: Signal Averaging Count perMeasurement: 128, Signal Accumulation Count: 3, and Interval: 2 seconds.

Prior to measurement, the system was blanked with isooctane. Each sampledispersion was measured 3 times and the mean values and the standarddeviation are reported. Data was evaluated using software WING SALD IIversion V3.1. Since the refractive index of the sample was unknown, therefractive index of sugar (disaccharide) particles (1.530) was used fordetermination of size distribution profiles. Size values for mean andmedian diameter are reported.

The mean particle sizes for all samples were very similar, slightlylower values were measured for HMO-Mix II. The particle sizecharacteristics are summarized in Table 5. In addition, the particlesize distribution showed the presence of one main size population forall of the samples.

TABLE 5 Particle size of HMOs as determined by laser diffraction Size3-fucosyllactose lacto-N-tetraose 6′-sialyllactose 3′-sialyllactoseHMO-Mix I HMO Mix II Mean 119.2 ± 0.5 117.3 ± 0.7 113.8 ± 1.5 115.4 ±0.6  113.1 ± 0.3  97.3 ± 5.3 [nm] Median 141.3 ± 0.0 141.3 ± 0.0 141.3 ±0.0 121.9 ± 16.7 141.3 ± 0.0 112.2 ± 0.0 [nm]

1. A spray-dried powder consisting essentially of LNT and/or LNnT whichhas been produced by microbial fermentation.
 2. The spray-dried powderaccording to claim 1, wherein the spray-dried powder comprises at least80%-wt., at least 85%-wt., at least 90%-wt., at least 93%-wt., at least95%-wt. or at least 98%-wt. LNT and/or LNnT.
 3. The spray-dried powderaccording to claim 1, wherein the LNT and/or LNnT is present inamorphous form.
 4. The spray-dried powder according to claim 1, whereinthe spray-dried powder comprises ≤15%-wt. of water, optionally ≤10%-wt.of water, optionally ≤7%-wt. of water, optionally ≤5%-wt. of water. 5.The spray-dried powder according to claim 1, wherein the spray-driedpowder is free of genetically-engineered microorganisms and nucleic acidmolecules derived from genetically-engineered microorganisms.
 6. Aprocess for the manufacture of a spray-dried powder as defined in claim1, the process comprising: a) purifying the LNT and/or LNnT from afermentation broth; b) providing an aqueous solution containing the LNTand/or LNnT of a); and c) subjecting the solution of b) to spray-drying.7. The process according to claim 6, wherein the purifying the LNTand/or LNnT from a fermentation broth (a)) includes one or more of i)removing the microbial cells from the fermentation broth to obtain acleared process stream; ii) subjecting the cleared process stream to atleast one ultrafiltration; iii) treating the cleared process stream atleast one time with a cation exchange resin and/or at least one timewith an anion exchange resin; iv) subjecting the cleared process streamto at least one nanofiltration and/or diafiltration; v) subjecting thecleared process stream to at least one electrodialysis; vi) treating thecleared process stream at least one time with activated charcoal; and/orvii) subjecting the cleared process stream at least one time to acrystallization and/or precipitation.
 8. The process according to claim6, wherein the aqueous solution comprises the LNT and/or LNnT in anamount of at least 20% (w/v), 30% (w/v), 35% (w/v), and up to 45% (w/v),50% (w/v), 60% (w/v).
 9. The process according to claim 6, wherein theaqueous solution comprising LNT and/or LNnT is spray-dried at a nozzletemperature of at least 110° C., optionally at least 120° C., optionallyat least 125° C., and less than 150° C., optionally less than 140° C.and optionally less than 135° C.
 10. The process according to claim 6,wherein the aqueous solution comprising LNT and/or LNnT is spray-driedat an outlet temperature of at least 60° C., optionally at least 65° C.,and less than 80° C., optionally less than 70° C.
 11. A productcomprising the spray-dried powder according to claim 1 for themanufacture of a nutritional composition, optionally an infant formula.12. A nutritional composition comprising the spray-dried powderaccording to claim
 1. 13. The nutritional composition according to claim12, further comprising at least one additional HMO, wherein said atleast one additional HMO is a neutral HMO or a sialylated HMO.
 14. Thenutritional composition according to claim 12, wherein the at least oneneutral HMO is selected from the group consisting of 2′-fucosyl lactose,3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose andlacto-N-fucopentaose I.
 15. The nutritional composition according toclaim 12, wherein the at least one sialylated HMO is selected from thegroup consisting of 3′-sialyllactose, 6′-sialyllactose,sialyllacto-N-tetraose (LST)-a, LST-b, LST-c anddisialyllacto-N-tetraose.
 16. The nutritional composition according toclaim 12, wherein the nutritional composition comprises at least oneprobiotic microorganism.